1. Field of the Invention
The present invention relates to a measuring apparatus, utilizing an evanescent wave, which analyzes a sample by causing a light beam to reflect at the interface between a thin film layer in contact with the sample and a dielectric block portion to generate an evanescent wave and then measuring a change in the intensity of the totally reflected light beam due to the evanescent wave.
2. Description of the Related Art
If free electrons vibrate collectively in a metal, a compression wave called a plasma wave will be generated. The compression wave, generated in the metal surface and quantized, is called a surface plasmon.
There are various kinds of surface plasmon resonance measuring apparatuses for quantitatively analyzing a substance in a liquid sample by taking advantage of a phenomenon that the surface plasmon is excited by a light wave. Among such apparatuses, one employing the “Kretschmann configuration” is particularly well known (e.g., see Japanese Unexamined Patent Publication No. 6(1994)-167443).
The surface plasmon resonance measuring apparatus employing the aforementioned “Kretschmann configuration” is constructed basically of (1) a dielectric block portion formed into the shape of a prism; (2) a metal film, formed on one surface of the dielectric block portion, for placing a measurement substance (which is a substance to measured) such as a liquid sample thereon; (3) a light source for emitting a light beam; (4) an optical system for making the light beam enter the dielectric block portion at various angles of incidence so that a condition for total internal reflection is satisfied at the interface between the dielectric block portion and the metal film; and (5) photodetection means for detecting the state of surface plasmon resonance (SPR), that is, state of attenuated total reflection (ATR) by measuring the intensity of the light beam totally reflected at the interface; and (6) measurement means for measuring the state of surface plasmon resonance (SPR) on the basis of the result of detection obtained by the photodetection means.
In order to obtain various angles of incidence in the aforementioned manner, a relatively thin light beam may be caused to strike the above-described interface at various incidence angles, or a relatively thick light beam may be caused to strike the interface convergently or divergently so that it has incident components at various angles. In the former, a light beam whose reflection angle varies with a change in the incidence angle can be detected by a small photodetector movable in synchronization with a change in the reflection angle, or by an area sensor extending in the direction where the reflection angle varies. In the latter, on the other hand, light beams reflected at various angles can be detected by an area sensor extending in a direction where the reflected light beams can be all received.
In the above-described surface plasmon resonance measuring apparatus, if a light beam strikes a metal film at a specific incidence angle θsp greater than a critical incidence angle at which total internal reflection (TIR) takes place, an evanescent wave having electric field distribution is generated in a measurement substance (liquid sample to be measured) in contact with the metal film. This evanescent wave excites the above-described surface plasmon in the interface between the metal film and the measurement substance (liquid sample to be measured). When the wave number vector of the evanescent wave is equal to the wave number of the surface plasmon and therefore the wave numbers between the two are matched, the evanescent wave resonates with the surface plasmon and the light energy is transferred to the surface plasmon. As a result, the intensity of the light totally reflected at the interface between the dielectric block portion and the metal film drops sharply. This sharp intensity drop is generally detected as a dark line by the above-described photodetection means.
Note that the aforementioned resonance occurs only when an incident light beam is p-polarized light. Therefore, it is necessary to make settings in advance so that an incident light beam strikes the aforementioned interface as p-polarized light.
If the wave number of the surface plasmon is found from an incidence angle θsp at which attenuated total reflection (ATR) takes place (the incidence angle θsp will hereinafter be referred to as a total reflection attenuation angle θsp), the dielectric constant of a measurement substance (liquid sample) can be calculated by the following Equation:
            K              s        ⁢                                  ⁢        p              ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              where Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in vacuum, and ∈m and ∈s represent the dielectric constants of the metal and the measurement substance, respectively.
That is, the properties related to the refractive index, can be found by finding the total reflection attenuation angle θsp which is an incidence angle at which the intensity of reflected light reduces
In this kind of surface plasmon resonance measuring apparatus, a photodiode array (photodetection means) can be employed with the object of measuring the aforementioned total reflection attenuation angle θsp accurately in a large dynamic range, as disclosed in U.S. Pat. No. 6,577,396. The photodetection means is constructed of a plurality of light-receiving elements juxtaposed in a predetermined direction. The light-receiving elements are juxtaposed to respectively receive the components of a light beam totally reflected at the aforementioned interface at various reflection angles.
In that case, there is provided differentiation means to differentiate optical detection signals output by the light-receiving elements of the aforementioned photodetection means, in the direction where the light-receiving elements are juxtaposed. Based on differentiated values output by this differentiation means, the total reflection attenuation angle θsp is specified, whereby the properties related to the refractive index of a measurement substance are often analyzed.
In addition, a leaky mode measuring apparatus is known as a similar measuring apparatus making use of an evanescent wave (for example, see “Spectral Researches,” Vol. 47, No. 1 (1998), pp. 21 to 23 and pp. 26 to 27). This leaky mode measuring apparatus consists basically of (1) a dielectric block portion formed into the shape of a prism; (2) a cladding layer formed on one surface of the dielectric block portion; (3) an optical waveguide layer, formed on the cladding layer, for placing a liquid sample thereon; (4) a light source for emitting a light beam; (5) an optical system for making the light beam enter the dielectric block portion at various angles of incidence so that a condition for total internal reflection is satisfied at the interface between the dielectric block portion and the cladding layer; and (6) photodetection means for detecting the excited state of a waveguide mode, that is, state of attenuated total reflection (ATR) by measuring the intensity of the light beam totally reflected at the above-described interface.
In the above-described leaky mode measuring apparatus, if a light beam strikes the cladding layer through the dielectric block portion at an incidence angle greater than a critical incidence angle at which total internal reflection (TIR) takes place, the light beam is transmitted through the cladding layer. Thereafter, in the optical waveguide layer formed on the cladding layer, only light with a specific wave number, incident at a specific incidence angle, propagates in a waveguide mode. If the waveguide mode is excited in this manner, most of the incident light is confined within the optical waveguide layer, and consequently, attenuated total reflection (ATR) occurs in which the intensity of light totally reflected at the aforementioned interface drops sharply. And the wave number of the light propagating through the optical waveguide layer depends upon the refractive index of the measurement substance (liquid sample) on the optical waveguide layer. Therefore, by finding the total reflection attenuation angle θsp at which attenuated total reflection ATR occurs, the refractive index of the measurement substance and the properties of the measurement substance related to the refractive index can be analyzed.
Note that the leaky mode measuring apparatus can also employ the aforementioned photodetection means (photodiode array) to detect the position of a dark line that occurs in reflected light because of attenuated total reflection (ATR) Also, in many cases, in addition to the photodetection means, the aforementioned differentiation means is employed in the leaky mode measuring apparatus.
In the field of pharmaceutical research, the above-described surface plasmon resonance measuring apparatus and leaky mode measuring apparatus are sometimes used in a random screening method for detecting a specific substance that couples to a sensing substance that is desired. In this case, a sensing substance is fixed as the above-described measurement substance on the aforementioned thin film layer (which is the aforementioned metal film in the case of surface plasmon resonance measuring apparatuses, or the cladding layer and optical waveguide layer in the case of leaky mode measuring apparatuses). Then, a liquid sample containing various inspection substances (which are substances to be inspected) is added to the sensing substance. And each time a predetermined time elapses, the total reflection attenuation angle θsp is measured.
If an inspection substance in the liquid sample is a substance that couple to the sensing substance, then the coupling will cause the refractive index of the sensing substance to vary with the lapse of time. Therefore, every time a predetermined time elapses, the total reflection attenuation angle θsp is measured. Based on the measured value, it is measured whether or not a change has occurred in the total reflection attenuation angle θsp. Based on this result, it can be judged whether or not the inspection substance is a specific substance that couples with the sensing substance. Examples of such a combination of a specific substance and a sensing substance are a combination of an antigen and an antibody, and a combination of an antibody and an antibody. More specifically, a rabbit antihuman IgG antibody and a human IgG (immunoglobulin G) antibody can be used as a sensing substance (which is fixed on a thin film layer) and a specific substance, respectively.
Note that in order to measure the coupled state between an inspection substance in a liquid sample and a sensing substance, the total reflection attenuation angle θsp itself does not always need to be detected. For example, a liquid sample with a target substance is added to a sensing substance. Next, a change in the total reflection attenuation angle θsp is measured. Based on the magnitude of the change, the coupled state between the inspection substance and the sensing substance can be measured. In the case where the aforementioned photodetection means and differentiation means are employed in a measuring apparatus utilizing ATR, a quantity of change in a differentiated value corresponds to a quantity of change in the total reflection attenuation angle θsp. Therefore, based on a quantity of change in a differentiated value, the coupled state between the sensing substance and the target substance can be measured (see Japanese Unexamined Patent Publication No. 2003-172694).
In the above-described measuring method and apparatus that utilize ATR, a liquid sample consisting of a solvent and an inspection substance is supplied to a cup-shaped or Petri dish-shaped measuring chip in which a sensing substance is fixed on a thin film layer formed on the bottom surface, and the above-described quantity of change in the total reflection attenuation angle θsp is measured.
Note that in Japanese Unexamined Patent Publication No. 2001-330560, there is disclosed a measuring apparatus, utilizing ATR, which is capable of measuring a great number of samples in a short time by serially measuring a plurality of measuring chips mounted in a turntable, etc.
In U.S. Patent Laid-Open No 20020046992, there is also disclosed a measuring apparatus, utilizing ATR, which performs measurements, employing a measuring chip provided with a plurality of sample-holding portions. In such a measuring apparatus, a great number of samples can be measured in a short time without moving the measuring chip.
In the above-described conventional measuring apparatuses, incidentally, a difference between adjacent light-receiving elements in the above-described photodetection means is generally computed by differentiation means and is output as a differentiated value. However, there are cases where there is an individual difference between the sensitivities of the light-receiving elements or cases where signals from the light-receiving elements undergo various noise or waveform distortion. In such a case, for example, a differentiated value, which should decrease and increase before and after a dark line according to an increase in an incidence angle θ, increases and then decreases. That is, a differentiated value does not vary linearly with a change in the incidence angle θ, and consequently, there is a possibility that accuracy in measuring the state of attenuated total reflection (ART) will degrade.